Project Summary The implementation of checkpoint blockade immunotherapy is having a major impact on cancer outcomes. However, a sizable subset of patients still fails to benefit, and prior research has shown that inadequate baseline T cell infiltration into the tumor microenvironment is associated with lack of clinical response. These findings point to poor spontaneous T cell activation and recruitment as a general mechanism of resistance and motivate the search for mechanisms that regulate the degree of T cell infiltration into the tumor microenvironment. Our group has previously identified tumor- intrinsic oncogene pathways as key variables that contribute to poor T cell recruitment and therapeutic resistance. Host-intrinsic factors such as germline polymorphisms in immune regulatory genes can also influence the degree of T cell infiltration. But in addition, a major environmentally-influenced variable has recently emerged, which is the composition of the commensal microbiota. Recent work in our group has identified the commensal microbiota as a key determinant of anti-tumor immunity and immunotherapy efficacy in a mouse melanoma model (Sivan et al. Science 2015). Based on that work, we pursued a similar correlation in human melanoma patients undergoing anti-PD-1 immunotherapy and found a profound correlation between baseline microbiota composition and clinical response, which was a significant biomarker for predicting treatment outcome (Matson et al. Science 2015). We further demonstrated a deeper causal link by colonizing germ-free mice with responder or non- responder patient microbiomes, which recapitulated the strong or poor therapeutic response to PD-1 blockade, which correlated with the degree of spontanaoues T cell priming against the tumor. In the current proposal, we will employ this model system to further pursue the mechanisms by which the commensal microbiota from humans modulates the anti-tumor immune response in vivo. A major goal is to identify the gut microbiota-driven messenger(s) capable of transmitting the immunomodulatory effect to the tumor site. Understanding these deeper mechanisms of immune potentiation could enable development of drugs that mimic the presence of immune-potentiating bacteria. Another major goal is to isolate and culture the human-derived commensal bacteria with immune-potentiating properties. This effort could lead to the development of probiotics to supplement immunotherapy regimens and improve outcomes in the future.